The desirability of modifying biologically active and therapeutically useful polypeptides with a variety of compounds having amine reactive groups, such as hydrophilic polymers, e.g., polyethylene glycol (PEG), to enhance their pharmacokinetic properties has been noted. See, e.g., the discussion of the art in this area of polypeptide modification in published PCT patent application No. WO87/00056. Such modification has been attempted to reduce adverse immune response to the polypeptide, increase the solubility for use in pharmaceutical preparations, and maintain a desirable circulatory level of such polypeptide for therapeutic efficacy.
One significant problem not addressed by the extensive art in this area of polypeptide modification involves the extent to which a polypeptide can be modified by attachment of compounds having amine reactive groups. For example, treatment of a polypeptide with PEG or similar polymers, can result in random attachment of the polymer at the amino terminus of the polypeptide and/or at one or more lysine residues in the amino acid sequence of the protein. While several PEG groups can attach to the polypeptide, the end result is a composition containing or potentially containing a variety of species of "PEG-ylated" polypeptide. Such heterogeneiety in composition is undesirable for pharmaceutical use.
The attachment of compounds with amine reactive groups to a polypeptide may alter the biological activity of the polypeptide. This effect is believed mediated by the position and number of the attachment site(s) along the polypeptide sequence. There thus remains in the art a need for a method enabling site specific attachment of such compounds to polypeptides, in a manner that enables the manipulation of the number and position of attachment sites. Such site specific attachments can generate homogeneously modified polypeptides which are therapeutically efficacious and which retain certain desirable characteristics of the natural polypeptides.